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Department of Cell and Molecular Biology Medical Nobel Institute Karolinska Institutet S-171 77 Stockholm, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The adenovirus E1A gene products have been shown to be multifunctional, playing central roles in the control of viral and cellular gene expression and transformation. Two structurally homologous proteins, of 243 and 289 amino acids (243R and 289R), are the major products encoded by E1A. E1A-289R contains three, among adenoviruses, highly conserved regions, designated conserved region (CR) 1, 2, and 3. These regions are important for the multiple activities ascribed to E1A. The major E1A transcription activation domain is contained within CR3 and is unique to E1A-289R. This domain participates in transcriptional activation by physically interacting with both basal and upstream binding transcription factors (13).
E1A has been shown to directly interact with retinoic acid receptor ß (RARß) and thereby function as a cofactor for activation of the RARß2 promoter (14). In cotransfection experiments, efficient activation of RARß2 by E1A and RARß also requires the addition of the TATA binding protein (TBP) (15). Furthermore, E1A has been shown to directly bind to TBP (16, 17, 18). These results suggest that E1A-289R mediates transcriptional activation by providing a physical bridge between TBP and RARß.
Retinoids play an important role in differentiation of embryonic carcinoma (EC) cells. Undifferentiated murine P19 EC cells differentiate into neurons, astrocytes, and fibroblast-like cells after addition of retinoic acid (RA) (19). An early and essential step in the differentiation process is the activation of the RARß2-promoter. However, activation of the RARß2-promoter in P19 cells does not require the viral E1A protein; instead, an endogenous E1A-like activity (E1A-LA) is used as a bridging factor between RARß and TBP (15). The existence of E1A-LA was first suggested when nonviable adenovirus E1A mutants were able to grow in undifferentiated EC cells (20).
Since RAR uses E1A/E1A-LA as a cofactor, we investigated whether
TR and E1A/E1A-LA also cooperate in transcriptional activation. In this
paper, we show that E1A and TR synergistically activate transcription
of promoters containing TR recognition sites. This activation is seen
for three different TR recognition elements [DR4>IR0>
-myosin
heavy chain (MHC)] and is mediated by all the isoforms of chicken
TRs tested.
The synergistic activation is accomplished through a direct interaction between TR and E1A. We have mapped the interacting domains of E1A to CR1 and CR3, both of which are also required for efficient enhancement of TR-mediated transcription. E1A interacts with the LBD and DBD of TR. The results presented here indicate that a complex containing TR and E1A is formed on a promoter containing a DR4 type of cis-acting element and that this complex is of sufficient importance to gain efficient transcriptional activation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-p46 were cotransfected
along with a chloramphenicol acetyltransferase (CAT)-reporter construct
(pBLCAT-TRE) containing a TR-responsive element into human chorion
carcinoma cells (JEG). These cells contain low endogenous TR (Fig. 1A
, right) (21) and no
endogenous E1A-like activity. We first studied the effects of E1A on
thyroid-mediated transcription via a direct repeat separated by four
nucleotides, since this is the the most commonly found TRE. Figure 1A
shows that E1A increased the level of TR-mediated transcription at
least 40-fold. As shown previously, TR alone activated transcription
3-fold via the single DR4 element in the presence of T3
(21). It is noteworthy that the basal activity in the absence of
T3 was also several fold increased by E1A, although not to
the same extent as when the ligand was added, 11- and 40-fold,
respectively (Fig. 1). When E1A was transfected without exogenous TR, a
small amount of T3-induced transcription was seen on all
elements tested (Fig. 1). This is most likely explained by interaction
of E1A with endogenous expressed TR in the JEG cells (21).
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MHC gene, which is normally expressed in heart muscle cells.
This element consists of two half-sites oriented as direct (DR4) and
palindromic (IR2) repeats separated by four and two nucleotides,
respectively. E1A was shown to enhance the TR-mediated activation
approximately 40-fold both via the heart-specific element and via a
palindromic (IR0) TR-responsive element (Fig. 1B
). Neither TR nor E1A could activate transcription of the corresponding control reporter plasmid containing only the RSV (Rous sarcoma virus) TATA box in front of the CAT-gene (pBLCAT), thus verifying that the activation obtained on the various TREs was TR and DNA specific.
The ubiquitously expressed RXR is known to heterodimerize with TR and
thereby increase transactivation. However, exogenous RXR had no
additional effect on the E1A-enhanced TR-mediated activation in our
cotransfection studies (Fig. 1A). Endogenous RXRs are present in low
amounts in JEG cells as shown by Wahlström et al.
(21).
During an adenovirus infection E1A efficiently activates transcription of the adenoviral E4 and E1A promoters. Therefore, the influence of TR on E1A- regulated transcription was assayed using these promoters as reporter constructs in cotransfections. Addition of TR had no effect on the activation by E1A (data not shown).
Different Isoforms of TR Cooperate with E1A in Transactivation
Several variants of the chicken thyroid hormone receptor
that are expressed in different tissues and at different developmental
times have been described (22, 23, 24, 25). The receptors differ in their N
termini, regions that have been shown to contain putative
phosphorylation sites and nuclear localization signals (26). We
therefore investigated whether the synergism between E1A and cTR was
TR isoform specific. Cotransfection experiments were done in the
presence of either TR-p46, TR-p40, TRß0, or TRß2 in the
absence or presence of E1A and T3 hormone (Fig. 2). Figure 2B shows that the synergistic
effect was obtained with all the chicken thyroid hormone receptors,
tested on a DR4 type of DNA element. These different receptors have
previously been shown to bind equally well to DR4 half-sites in
vitro (Ref. 3 and data not shown). Interestingly, the N-terminal
shorter variants (TR-p40 and TRß0) gave a slightly lower level of
activation.
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To investigate how the CR1, CR2, and CR3 domains of the E1A protein
exert their effects on TR-mediated activation, we transfected plasmids
encoding E1A deletion mutants along with TR into JEG cells. Figure 3 shows that the 
CR3 mutant activated
transcription to 30% of the wild-type (wt) E1A protein, indicating
that the E1A-enhanced activation is mainly dependent on this domain. A
mutant lacking CR1 activated TR-regulated transcription to 50% of
E1Awt, and the mutant lacking CR2 enhanced the transactivation to the
same extent as E1Awt, whereas a double mutant of CR1 and CR3 gave, as
expected, the lowest transactivation. Taken together, preferentially
CR3, but also CR1, contributes to E1A enhancement of TR-mediated
transactivation in JEG cells.
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shows that the wt E1A
protein strongly bound to GST-TR, both in the presence and absence of
T3. As expected, in vitro translated RXR was
also bound by GST-TR. The E1A carboxy-terminal binding protein, CtBP
(31), here used as a negative control, did not bind efficently to
GST-TR compared with input levels. The binding of the E1A 289-amino
acid protein to GST-TR was strong since E1A still bound efficiently in
high-salt buffer (500 mM NaCl, data not shown).
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).
As the E1A 1–130 and E1A 1–89 proteins both lack CR3, the result
suggests that CR3 is important for efficient binding to TR.
An E1A mutant containing both CR1 and CR3, but lacking the
carboxy-terminal amino acids 193–245, was retained by GST-TR at the
same efficiency as the wt E1A protein (Fig. 4C). This deletion spans
the AR1 region that is required for activation of the E4F transcription
factor (32).
The E1A mutant with an internal deletion of CR1 bound to GST-TR,
although to a lesser extent than the wt protein (Fig. 4D). The mutant
with an internal deletion of CR3 also weakly bound to GST-TR (Fig. 4D).
The double mutant, lacking both CR1 and CR3, was totally deficient in
binding GST-TR. These results demonstrate that the CR1 and CR3 domains
of E1A cooperate to form a stable complex with TR.
The bacterially expressed GST-TR bound to the DR4 recognition sequence,
as determined by gel-shift analysis. The GST-TR protein was also
recognized by an -TR antibody in Western blots (data not shown).
This indicates that expression of TR in bacteria does not destroy its
conformation and function.
Domains of Interaction in TR
In a set of reciprocal experiments, different GST-E1A fusion
proteins were used to assay binding of in vitro translated
TR proteins. Both the TR variants, p46 and p40, bound to GST-E1A
containing either CR1 or CR3 (GST-E1ACR3 and GST-E1A76–289) (Fig. 5B). None of the TR proteins bound to the
GST-E1ACt mutant, which contains amino acids 200–243 of the
carboxy-terminal domain of E1A (Fig. 5B). This is in agreement with the
results presented above in Fig. 4. RAR bound to GST-E1A proteins
containing CR1 or CR3, whereas RXR did not associate with any of the
GST-E1A proteins. CtBP, now used as a positive control, was retained by
all E1A proteins, as they all have the carboxy-terminal domain
present.
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). The TR mutant encoding amino acids 49–408 bound efficiently
to GST-E1A 76–289 and GST-E1ACR3. This TR mutant contains both the
DBD and LBD. The p46 122–408 mutant, containing the LBD, also bound to
GST-E1A 76–289 and GST-E1ACR3, although not as strongly as p46
49–408 (Fig. 5C). The mutants p46 1–256, p46 49–256, and p46
49–118, all containing the DBD, bound weakly to GST-E1A 76–289, but
not to GST-E1ACR3 (Fig. 5, C and D). This suggests that there is a
weak interaction between TR DBD and E1A CR3.
A clear interaction was found between p46 257–408 and GSTE1A 76–289
(Fig. 5D). This result indicates that the main interactions are made
between the carboxy-terminal part of the LBD in TR and CR3 in E1A. The
p46 1–118 and p46 122–256 mutants were not retained by any of the
GST-E1A proteins.
The shorter amino terminus TR variant, p40, bound more efficiently to
GST-E1A than the full-length variant, p46 (Fig. 5B). Similarly, the
mutants p46 49–256 and p46 49–118 bound GST-E1A better than the
corresponding amino termini containing mutants p46 1–256 and p46
1–118, respectively (Fig. 5, C and D). In all these cases, addition of
the amino terminus to the TR mutant weakened its ability to bind to
E1A. This indicates that the amino terminus has a negative influence on
the interaction between TR and E1A.
In summary, the CR3 in E1A interacts with both the carboxy terminus and the DBD of TR, while CR1 in E1A makes contact with TR somewhere in the LBD. The strongest binding is seen when both the DBD and LBD are present in TR. In contrast, the amino terminus of TR has a negative effect on binding to E1A.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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E1A has been shown, in an analogous way, to interact with RARß, and thereby activate transcription of the RARß2 gene. The direct interaction between E1A and RARß was shown to depend on CR3 in E1A and the LBD including AF2 in RARß (14). In our study, two regions in E1A, CR1 and CR3, bound to TR, with stronger affinity for CR3. These two regions appear to cooperate to obtain an efficient binding. Our results from the binding experiments are in agreement with our cotransfection experiments, which demonstrated that a mutant containing CR3 gave 50% and a mutant containing CR1 gave 30% of the activation normally observed with E1Awt. In the study of Folkers and van der Saag (14), RARß-mediated transactivation was dependent on CR3. The observed differences between their study and the results presented in this paper may be explained by different requirements of E1A domains for TR- and RARß-induced transactivation and by the use of different cell lines.
The region in TR required for interaction with E1A is primarly the carboxy-terminal portion of the LBD, including AF-2. This is in agreement with the results of Folkers and van der Saag, as they have suggested that the region required for E1A binding in RARß is in the LBD. In addition, we also see a minor binding between E1A and the DBD of TR. Thus, we suggest that two separate regions in E1A interact with two separate regions in TR.
Several TR-interacting cofactors have been identified recently. Two of
these cofactors, p300/CBP and SRC-1, have been found to possess
intrinsic histone acetyltransferase activities; consequently, it was
suggested that these cofactors may remodel transcriptionally
repressed chromatin to make the promoter accessible for general
transcription factors (33, 34, 35, 36). Other cofactors are thought to enhance
and stabilize the assembly of the preinitiation complex. The latter is
likely one function of E1A, since it interacts with both upstream and
basal transcription factors. On the other hand, recent reports have
suggested that E1A may inhibit p300/CBP histone acetyltransferase
activity and also the transcription mediated by nuclear hormone
receptors. (37, 38). However, the histone acetyltransferase activity of
p300/CBP is necessary for transcriptional activation by the
transcription factor Stat1, but not for the nuclear retinoid
receptor RAR, (39, 40). Instead, E1A inhibits RAR transactivation by
preventing the association of CBP with nuclear receptor coactivators
(40). Taken together, the recent reports suggest that E1A activates
transcription of nuclear receptors when E1A interacts with the receptor
and thereby localizes to the promoter. Not juxtaposed to the promoter,
E1A would instead repress nuclear receptor-mediated transcription
through disassembling receptor-coactivator complexes. The ability of
E1A to function as a coactivator or a corepressor may be influenced by
the promoter context and by the composition of nuclear receptor dimers
as well as by cell type-specific cofactors.
What is the biological significance of an interaction between a nuclear receptor and the adenovirus E1A oncoprotein? As these two proteins can only coexist in adenovirus-infected cells, one function could be that the virus uses TR for modifying E1A-regulated gene transcription. However, since TR did not affect E1A-induced activation of the E1A- and E4-promoters, this is not likely to be the case. Instead, we hypothesize that the adenovirus E1A oncoprotein mimics the function of E1A-LA of undifferentiated EC cells, and that the interaction between TR and E1A-LA would be confined to early, pluripotent cells. E1A-LA would, in such a model, enhance the activating potential of TR on differentiation-specific genes. Our preliminary results suggest that activation of TR-driven transcription through DR4 sites is not further enhanced by the addition of viral E1A in undifferentiated P19 cells (data not shown). This indicates that E1A-LA could cooperate with TR to induce efficient transactivation in P19 cells. Furthermore, T3 treatment of P19 cells induces the cells to differentiate into cardiac myocytes (41). The authors suggest that this differentiation is accompanied by preferential binding of as yet undefined factors to DR4-sites (42). Whether or not these factors are TR and E1A-LA remains to be elucidated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 (p40
and p46) and TRß0 and ß2 receptors were cloned into the pSG5
expression vector (3, 43). The plasmid pML005, used for expressing
E1Awt in transfections, contains nucleotides 1–1773 of genomic
adenovirus type 2. The CR1, CR2, and CR3 mutants of pML005 has been
described previously (44). GSTE1A 76–289 and GSTE1ACR3 (GSTE1A12S)
have been described previously (45). GST-TR was cloned by inserting the
EcoRI fragment of pSG65-p46 into the EcoRI site
of the pGEX1lambdaT vector (Pharmacia Biotech, Piscataway,
NJ). Gst-E1ACt (aa 200–243) and pcDNA3-CtBP have been described
previously (31, 46). pML00512S, pML00513S (47), and pSG5p46 were used
as templates for making in vitro translated E1A and TR
proteins, respectively.
Transfections
Human chorion carcinoma cells (JEG) were plated at a density of
2 x 105 per 3-cm dish in DMEM (Biological
Industries) supplemented with 8% FCS. One day after plating,
the medium was replaced with DMEM containing 8% calf serum depleted of
T3 and T4 by ion exchange resin (48).
Approximately 2 h later the cells were cotransfected with
expression vectors encoding 100–200 ng of different chicken TRs, E1A,
or E1A mutants plus 500 ng of reporter constructs containing a TRE. The
cells were maintained in the presence or absence of 12–30
nM T3, harvested 24 h after hormone
treatment, and assayed for chloramphenicol acetyltransferase activity.
Quantifications were done with a Molecular Dynamics, Inc.
(Sunnyvale, CA) phosphorimager (49). All transfections were repeated at
least three times with similar results. Duplicate sample points were
used in each experiment and varied by less than 15%.
Protein Binding Analysis
In vitro GST-fusion proteins were produced in
Escherichia coli and purified with gluthathione agarose
beads (Current Protocols in Molecular Biology). Protein
concentrations were estimated on a Coomassie-stained SDS-polyacrylamide
gel. Approximately equal amounts of GST fusion proteins were mixed with
5–10 µl of [35S]methionine-labeled in vitro
synthesized proteins (TNT-coupled reticulocyte lysate systems,
Promega Corp., Madison, WI). The proteins were incubated
rotating at 4 C for 3 h in binding buffer (250–500 mM
NaCl, 50 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.1%
NP40, 1 mM dithiothreitol, and 0.2 mM
phenylmethylsulfonylfluoride). Beads were washed four times in
binding buffer, and bound proteins were separated on a polyacrylamide
gel and visualized by autoradiography. In Figs. 4D and 5, C and D, the
35S-labeled proteins were synthesized using PCR-amplified
DNA templates. The 5'-PCR primers contained the sequence for a T7 RNA
polymerase start site.
Western Blot
Equal amounts of crude extracts made from human chorion
carcinoma (JEG) and human cervical carcinoma (HeLa) cells were run on a
10% denatured gel. Separated proteins were transferred overnight to
enhanced chemiluminescence (ECL) nitrocellular membranes, which were
then hybridized to a primary rabbit polyclonal antibody directed
against TR diluted 1:1000 (FL-408, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After incubation with a secondary
peroxidase-conjugated goat antirabbit antibody diluted 1:1000
(DAKO Corp., Carpinteria, CA), the membranes were washed
and the migrated proteins were detected with the ECL system
(Amersham, Arlington Heights, IL).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This project was funded by grants from Cancerfonden, the Lars Hierta Foundation, the Magnus Bergvalls Foundation, Human Fronties, Karolinska Institutet, and the Swedish Society for Medical Research. M.B-B. was also supported by the Swedish Natural Science Research Council.
1 These authors have contributed equally to the work. 
Received for publication December 17, 1998. Revision received April 12, 1999. Accepted for publication April 16, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß thyroid
hormone receptor genes. EMBO J 9:1519–1528[Medline]
and N-terminal variant ß receptors. Development 114:39–47[Abstract]
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